Biology and biotechnology of Trichoderma

Fungi of the genus Trichoderma are soilborne, green-spored ascomycetes that can be found all over the world. They have been studied with respect to various characteristics and applications and are known as successful colonizers of their habitats, efficiently fighting their competitors. Once established, they launch their potent degradative machinery for decomposition of the often heterogeneous substrate at hand. Therefore, distribution and phylogeny, defense mechanisms, beneficial as well as deleterious interaction with hosts, enzyme production and secretion, sexual development, and response to environmental conditions such as nutrients and light have been studied in great detail with many species of this genus, thus rendering Trichoderma one of the best studied fungi with the genome of three species currently available. Efficient biocontrol strains of the genus are being developed as promising biological fungicides, and their weaponry for this function also includes secondary metabolites with potential applications as novel antibiotics. The cellulases produced by Trichoderma reesei, the biotechnological workhorse of the genus, are important industrial products, especially with respect to production of second generation biofuels from cellulosic waste. Genetic engineering not only led to significant improvements in industrial processes but also to intriguing insights into the biology of these fungi and is now complemented by the availability of a sexual cycle in T. reesei/Hypocrea jecorina, which significantly facilitates both industrial and basic research. This review aims to give a broad overview on the qualities and versatility of the best studied Trichoderma species and to highlight intriguing findings as well as promising applications

Role of the sympathetic nervous system in carbon tetrachloride-induced hepatotoxicity and systemic inflammation

Carbon tetrachloride (CCl4) is widely used as an animal model of hepatotoxicity and the mechanisms have been arduously studied, however, the contribution of the sympathetic nervous system (SNS) in CCl4-induced acute hepatotoxicity remains controversial. It is also known that either CCl4 or SNS can affect systemic inflammatory responses. The aim of this study was to establish the effect of chemical sympathectomy with 6-hydroxydopamine (6-OHDA) in a mouse model of CCl4-induced acute hepatotoxicity and systemic inflammatory response. Mice exposed to CCl4 or vehicle were pretreated with 6-OHDA or saline. The serum levels of aminotransferases and alkaline phosphatase in the CCl4-poisoning mice with sympathetic denervation were significantly lower than those without sympathetic denervation. With sympathetic denervation, hepatocellular necrosis and fat infiltration induced by CCl4 were greatly decreased. Sympathetic denervation significantly attenuated CCl4-induced lipid peroxidation in liver and serum. Acute CCl4 intoxication showed increased expression of inflammatory cytokines/chemokines [eotaxin-2/CCL24, Fas ligand, interleukin (IL)-1α, IL-6, IL-12p40p70, monocyte chemoattractant protein-1 (MCP-1/CCL2), and tumor necrosis factor-α (TNF-α)], as well as decreased expression of granulocyte colony-stimulating factor and keratinocyte-derived chemokine. The overexpressed levels of IL-1α, IL-6, IL-12p40p70, MCP-1/CCL2, and TNF-α were attenuated by sympathetic denervation. Pretreatment with dexamethasone significantly reduced CCl4-induced hepatic injury. Collectively, this study demonstrates that the SNS plays an important role in CCl4-induced acute hepatotoxicity and systemic inflammation and the effect may be connected with chemical- or drug-induced hepatotoxicity and circulating immune response

From an assessment of multiple chelators, clodronate has potential for use in continuous chelation

Aim: To identify chelators which when mixed with sodium hypochlorite (NaOCl) are stable, exhibiting minimal loss of free available chlorine (FAC) over 80 min and to further investigate potential mixtures by assessing FAC over 18 h and the capacity to remove smear layer. Methodology: 0.25 mol L−1 EDTA (10%), 0.25 mol L−1 EGTA (egtazic acid), 0.25 mol L−1 CDTA (cyclohexanediaminetetraacetic acid), 0.25 mol L−1 DTPA (pentetic acid), 0.5 mol L−1 ATMP (aminotri(methylene phosphonic acid)) and 1 mol L−1 HPAA, (hydroxyphosphonoacetic acid), all at alkaline pH, were mixed equally with 5% NaOCl. 0.5 mol L−1 alkaline clodronate and 0.5 mol L−1 Na4etidronate (15%) were mixed equally with 10% NaOCl. For all mixtures, the pH and temperature were measured over 80 min and additionally for the clodronate mixture over 18 h. Iodometric titration was used to measure the FAC of all mixtures except for HPAA. The following were compared with respect to their ability to remove smear layer: 1 mol L−1 clodronate + 10% NaOCl, 0.5 mol L−1 clodronate + 10% NaOCl, 1 mol L−1 etidronate + 10% NaOCl, 0.5 mol L−1 clodronate + 10% NaOCl and the sequences 5% NaOCl/17% EDTA/5% NaOCl and 5% NaOCl/17% EDTA. The area fraction occupied by open dentinal tubules as a percentage of the total area (% AF) from scanning electron microscopy micrographs was calculated using Image J. The results were statistically analysed with alpha set at 0.05. Results: Compared to its control, the mixture 0.5 mol L−1 clodronate + 10% NaOCl lost no FAC over 18 h (P > 0.05). The FAC of 0.25 mol L−1 CDTA mixed with 5% NaOCl fell to 96%, 92%, 75% and 4.9% at 20, 40, 60 and 80 min, respectively. Temperature rises were observed in all cases except in the etidronate and clodronate mixtures. Only in the clodronate mixture did the pH remain above pH 12 for the whole experiment. Although smear layer was removed, the % AF in 1 mol L−1 clodronate + 10% NaOCl, 0.5 mol L−1 clodronate + 10% NaOCl, 1 mol L−1 etidronate + 10% NaOCl was less than for 0.5 mol L−1 etidronate + 10% NaOCl and 5% NaOCl/17% EDTA/5% NaOCl and 5% NaOCl/17% EDTA. Conclusion: Alkaline 0.5 mol L−1 clodronate mixed equally with 10% NaOCl has potential for use in continuous chelation, based on this assessment of stability and smear layer removal. Further research is needed to establish its efficacy and safety

Low frequency of spontaneous rearrangements during plasmid incorporation in CHO-K1 mutant cells defective in DNA repair

pLrec plasmid DNA was introduced into Chinese hamster cell lines defective in DNA repair (Pa13, Pb4, xrs6) and the parental CHO-K1 cell line. Clones with stable integrated plasmid were isolated and integrity of the incorporated DNA was checked by Southern blotting and PCR. Intact pLrec plasmid was found in 11% of the isolated CHO-K1 clones. In contrast, intact plasmid copies were found in 68.8%, 50%, 35.7% clones of Pa13, Pb4 and xrs6 cell lines, respectively. We conclude that certain DNA repair defects may facilitate intact plasmid integration. The higher frequency of integration of the intact vector into the genome of cell lines defective in DNA repair as compared to the parental cell line points to two possibilities, not mutually exclusive: (1) these cells possess a mechanism that facilitates the process of plasmid incorporation and hence, plasmid DNA is incorporated into the genome before extrachromosomal recombination takes place; (2) the vector is inserted into a less recombination prone site in the genome